Lithographic mask design and synthesis of diverse probes on a substrate

ABSTRACT

Systems and methods of synthesizing probes on a substrate are provided. One or more shift reticles are utilized to uniformly add monomers to the substrate at specified locations. The shift reticles are shifted relative to the substrate between monomer addition steps. Additionally, characteristics of the desired probes may be specified at synthesis time.

This is a continuation-in-part of application Ser. No. 08/767,892, filedDec. 17, 1996, abandoned, which is hereby incorporated by reference forall purposes.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialthat is subject to copyright protection. The copyright owner has noobjection to the xerographic reproduction by anyone of the patentdocument or the patent disclosure in exactly the form it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

MICROFICHE APPENDIX

A Microfiche Appendix (1 microfiche, 72 frames) of a computer programlisting of an embodiment of the invention is included herewith.

BACKGROUND OF THE INVENTION

The present invention is related to computer systems for generatingmasks. More particularly, the invention provides systems and methods forgenerating and utilizing masks to form probes on a substrate.

U.S. Pat. No. 5,424,186 describes a pioneering technique for, amongother things, forming and using high density arrays of molecules such asoligonucleotide, RNA, peptides, polysaccharides, and other materials.This patent is hereby incorporated by reference for all purposes. Arraysof oligonucleotides or peptides, for example, are formed on the surfaceby sequentially removing a photoremovable group from a surface, couplinga monomer to the exposed region of the surface, and repeating theprocess. These techniques have been used to form extremely dense arraysof oligonucleotides, peptides, and other materials. Such arrays areuseful in, for example, drug development, oligonucleotide sequencing,oligonucleotide sequence checking, and a variety of other applications.The synthesis technology associated with this invention has come to beknown as "VLSIPS" or "Very Large Scale Immobilized Polymer Synthesis"technology.

Additional techniques for forming and using such arrays are described inU.S. Pat. No. 5,384,261, which is also incorporated by reference for allpurposes. Such techniques include systems for mechanically protectingportions of a substrate (or chip), and selectively deprotecting/couplingmaterials to the substrate. These techniques are now known as "VLSIPSII." Still further techniques for array synthesis are provided in U.S.application Ser. No. 08/327,512, also incorporated herein by referencefor all purposes.

Dense arrays fabricated according to these techniques are used, forexample, to screen the array of probes to determine which probe(s) arecomplementary to a target of interest. According to one specific aspectof the inventions described above, the array is exposed to a labeledtarget. The target may be labeled with a wide variety of materials, butan exemplary label is a fluorescein label. The array is then scannedwith a confocal microscope based detection system, or other relatedsystem, to identify where the target has bound to the array. Otherlabels include, but are not limited to, radioactive labels, largemolecule labels, and others.

While meeting with dramatic success, such methods meet with limitationsin some circumstances. For example, during the design of the layout ofmolecules in an array according to the above techniques, it is necessaryto design a "mask" that will define the locations on a substrate thatare exposed to light. While such masks are easily fabricated, they tendto be costly. The design of such masks is described in U.S. Pat. No.5,571,639, incorporated herein by reference for all purposes.

Often it is desirable to have a specific layout of molecules in an arrayfor a particular application. For example, PCT WO95/11995, which isincorporated by reference for all purposes, describes the synthesis ofparticular arrays for use in HIV diagnostics, the diagnosis of genesrelevant to certain cancers, evaluation of the mitochondrialoligonucleotide, and other applications. In many of these applicationsthere is demand for a large volume of identical chips, such as in HIVdiagnostics. In many situations, the manufacture of a particular probearray will require a mask (or mask set) with as many as one hundredreticles or more. The cost of masks in these situations, while high on aper mask basis, becomes quite small when viewed in light of the numberof identical arrays that may be synthesized with a particular mask.

However, in many other applications, such as particular researchapplications, it is desirable to synthesize a relatively small number ofarrays with a particular layout of probes, perhaps as few as a singlearray. While this is certainly possible and has found wide utility inthe art, it is costly to fabricate a single mask (or mask set) for themanufacture of only a few probe arrays. Accordingly, the "per chip" costof masks in these situations can be quite high (on the order ofthousands of dollars).

Accordingly, it is desirable to identify more efficient techniques fordesigning and using lithographic masks in the manufacture of probearrays and, in particular, reduce the number of reticles required for alow volume design.

SUMMARY OF THE INVENTION

The present invention provides techniques for more economicallysynthesizing arrays of probes on a substrate. One or more "shift"reticles are utilized to synthesize many different probe sets on asubstrate. A shift reticle is a reticle that is shifted (one position ormore) after a monomer addition step and then reused which reduces thenumber of reticles (or masks) required. Additionally, the shift masksuniformly add monomers to the substrate at certain probe locationsduring synthesis. Embodiments of the invention allow the length of theprobes and interrogation position to be specified at synthesis timethereby providing greater flexibility in chip synthesis.

In one embodiment of the invention, a method of synthesizing probes on asubstrate, comprises the steps of: coupling monomers on the substrate atlocations specified by at least one shift reticle; shifting the at leastone shift reticle relative to the substrate; and after shifting the atleast one shift reticle, coupling monomers on the substrate at locationsspecified by the at least one shift reticle; wherein probes includingmonomers are synthesized on the substrate.

In another embodiment of the invention, a method of synthesizing probeson a substrate, comprises the steps of: providing at least one reticle,the at least one reticle for uniformly adding monomers to the substrateat specified locations; receiving input as to a characteristic of theprobes desired; and utilizing the at least one reticle to synthesize thedesired probes on the substrate. The characteristic may be the length ofthe desired probes, the interrogation position, or the monomer additionorder for synthesizing the desired probes.

In another embodiment of the invention, a method of synthesizing probeson a substrate comprises the steps of: providing a set of reticleshaving monomer addition regions, each reticle for coupling a differenttype of monomer on the substrate; utilizing each reticle of the set tocouple a first layer of monomers on the substrate, the first layer ofmonomers including different types of monomers; and shifting eachreticle of the set relative to the substrate to couple a second layer ofmonomers on the first layer, the second layer of monomers includingdifferent types of monomers; wherein a plurality of probes including twomonomers are formed on the substrate.

In another embodiment, a method for determining the layout of a reticlefor synthesizing probes on a substrate comprises the steps of: receivinginput of a target sequence of monomers; selecting a type of monomer inthe target sequence; and designing a reticle with monomer additionregions corresponding to each monomer in the target sequence that is theselected type of monomer.

In another embodiment, a method of synthesizing probes on a substratecomprises the steps of: coupling a plurality of first monomers on thesubstrate at locations specified by a set of monomer addition regions ofa reticle; shifting the reticle relative to the substrate; and couplingat least one second monomer on one of the first monomers at a locationspecified by one of the set of monomer addition regions of the reticle;wherein a probe including the first and second monomers is formed on thesubstrate.

In another embodiment, a computer-implemented method for determining thelayout of a reticle for synthesizing probes on a substrate comprises thesteps of: receiving input of a target sequence of monomers; selecting atype of monomer in the target sequence; and designing a reticle withmonomer addition regions specified by

    n*(i-1)+1

wherein n= the number of different types of monomers and i= a positionof a first monomer in the target sequence.

In another embodiment, a method for specifying the layout of a substrateincluding probes synthesized on the substrate, comprises the steps of:defining the probes to be synthesized on the substrate as a sequentiallist of analysis regions, each analysis region including probes;receiving input as to a characteristic of the sequential list ofanalysis regions; and designing at least one reticle to synthesize theprobes of each analysis region on the substrate with the inputcharacteristic. Typically the input characteristic includes thelocation, scale or orientation of the analysis regions.

In another embodiment, a method of synthesizing rows of probes includingan interrogation position on a substrate, comprising the steps of:coupling non-interrogation position monomers on the substrate in a firstregion having a first width; and coupling rows of interrogation positionmonomers on the substrate in a second region having a second width, thesecond region being within the first region and the second width beingless than the first width. Hybridization data from the probes may bemore accurate because the "edge effect" between adjacent probe regionsor cells is reduced.

In other embodiments, shift masks may be utilized to synthesize diverseprobes for interrogating a base position in a target. For example,probes of a specific length may be synthesized that include everypossible interrogation position in the probes. Additionally, probes ofdifferent lengths with different interrogation positions may besynthesized on a chip at the same time.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of a computer system used to executesoftware embodiments of the present invention;

FIG. 2 shows a system block diagram of a typical computer system used toexecute software embodiments of the present invention;

FIG. 3A illustrates a probe set that may be utilized to detect a targetsequence and FIG. 3B shows a layout of the probe set on a substrate inone embodiment;

FIG. 4 shows prior art reticles that produce the probe set of FIG. 3A;

FIG. 5 shows the prior art addition of monomers to produce the probe setof FIG. 3A;

FIG. 6 shows a high level flow of a process of generating reticlesaccording to one embodiment of the present invention;

FIG. 7 shows shift reticles that produce the probe set of FIG. 3A;

FIGS. 8A-8C shows the addition of monomers using the shift reticles toproduce the probe set;

FIGS. 9A-9B show reticles for producing multiple probe sets;

FIG. 10 shows the transformation of a linear reticle into a rectangularreticle;

FIG. 11A shows a reticle for adding monomers at an interrogationposition; FIG. 11B shows probes on a chip that vary at an interrogationposition; and FIG. 11C is an image of a chip produced with a reticlesimilar to the one in FIG. 11A;

FIG. 12A shows another reticle for adding monomers at an interrogationposition;

FIG. 12B shows probes on a chip that vary at an interrogation position;and FIG. 12C is an image of a chip produced with a reticle similar tothe one in FIG. 12A;

FIG. 13A shows a reticle for producing multiple probe set of differentlengths and FIG. 13B shows a chip with multiple length probe sets;

FIG. 14A is a mask including multiple reticles; FIG. 14B shows thelayout of a reticle in one embodiment; FIG. 14C shows reticles forsynthesizing probes on two chips simultaneously; FIG. 14D shows a maskfor synthesizing varying length probes on two chips simultaneously; andFIG. 14E shows a sample chip layout;

FIG. 15A shows a layout of a chip in another embodiment; FIG. 15B showsa shift reticle for coupling a particular monomer on a chip in pairs ofrows; FIG. 15C shows a shift reticle for coupling a particular monomeron a chip in a single lane; and FIG. 15D shows a shift reticle forforming control lanes;

FIG. 16 shows a high level flow of a process of generating reticlesaccording to another embodiment of the present invention;

FIGS. 17A-17D show the formation of a single shift reticle;

FIG. 18 shows a single reticle that produces the probe set of FIG. 3Aand the addition of monomers using this reticle;

FIG. 19 shows a reticle for producing multiple probe sets;

FIG. 20A shows a shift reticle for another embodiment;

FIGS. 20B-20D shows interrogation position reticles; and

FIG. 20E shows a chip including perfectly complementary, interrogationposition and deletion probes;

FIG. 21A shows a shift reticle for synthesizing related probes ofvarying lengths and FIG. 21B shows an example of the layout of theprobes that may be synthesized on the substrate;

FIG. 22 is a simple example of speckle masks;

FIG. 23 shows that the shift reticles of FIG. 7 are speckle masks;

FIG. 24 shows the packing of speckle masks;

FIG. 25 shows the layout of a chip utilizing post chip synthesis;

FIG. 26 shows the layout of another chip utilizing post chip synthesis;

FIG. 27A shows an active region of a chip that has tightly packed lanes;FIG. 27B shows a subregion from FIG. 27A; and FIG. 27C shows how thesubregion of FIG. 27B may be synthesized to minimize edges;

FIG. 28 shows shift reticles that produce equal length probes withdifferent interrogation positions;

FIG. 29 illustrates the probes that may be produced by the reticles ofFIG. 28;

FIG. 30 shows a shift reticle for producing probes with differentlengths and interrogation positions;

FIG. 31 illustrates the probes that may be produced by the shiftreticles according to FIG. 30; and

FIG. 32 shows a shift reticle for producing probes that interrogateevery ninth base position in a target.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Terminology

As used herein, the following terms are intended to have the followingmeanings:

"Mask" refers to a lithographic member, usually a plate of glass, with anumber of apertures therein that allow for selective passage of light. Amask may contain one or more reticles.

"Reticle" refers to all or a particular portion of a mask that is usedto direct light to a substrate during an exposure.

Introduction

High density, miniaturized arrays of molecular probes are made hereinusing light directed synthesis techniques. Such arrays may be arrays ofoligonucleotides, peptides, small molecules (such as benzodiazapines,prostoglandins, beta-turn mimetics), non-natural ligands, enzymes, orany of a wide variety of other molecules synthesized in a "buildingblock" fashion, such as oligosaccharides, and the like. Oligonucleotideprobe arrays are representative of the arrays that may be used accordingto specific aspects of the invention herein.

The design and fabrication of oligonucleotide probe arrays relies onVLSIPS technology according to a specific aspect of the invention. Thefirst step in fabricating a oligonucleotide probe array involveschoosing a set of oligonucleotide probes to be synthesized on the chip.Suppose, for example, it is desirable to detect a base change mutationat a single position in gene. The techniques herein would provide a setof four probes that are complementary to a short region around thesingle position. The first probe would be exactly complementary to thewild-type (normal) sequence for that region of the gene. The other threeprobes would be identical to the first, except they would substitute thethree bases that are not complementary to the wild-type sequence at theposition being interrogated (i.e., the interrogation position).

In this way regardless of the base change mutation, one of the probeswill be perfectly complementary to the target oligonucleotide sequence.To detect any such mutation in the gene, i.e., to resequence the gene,one may define similar sets of probes for each position in the gene. Forexample, to resequence the 1040 bases of HIV necessary to detect drugresistance related mutations, 4160 probes are generally required. Suchtechniques are described in greater detail in PCT WO95/11995 which isincorporated herein by reference for all purposes. Of course, arrayssuch as peptide arrays will provide for different techniques of probeselection.

Once a set of probes is chosen, the layout of the probes on the chip isdetermined. The layout is used to design the photolithographic masksused in chip synthesis process. These designs in general are produced inelectronic form and are used to fabricate the masks in a maskfabrication shop such as those widely used in the semiconductorindustry.

FIG. 1 illustrates an example of a computer system used to executesoftware embodiments of the present invention to generate masks for chipsynthesis. FIG. 1 shows a computer system 1 which includes a monitor 3,screen 5, cabinet 7, keyboard 9, and mouse 11. Mouse 11 may have one ormore buttons such as mouse buttons 13. Cabinet 7 houses a CD-ROM drive15 and a hard drive (not shown) that may be utilized to store andretrieve software programs including computer code incorporating thepresent invention. Although a CD-ROM 17 is shown as the computerreadable medium, other computer readable media including floppy disks,DRAM, hard drives, flash memory, tape, and the like may be utilized.Cabinet 7 also houses familiar computer components (not shown) such as aprocessor, memory, and the like.

FIG. 2 shows a system block diagram of computer system 1 used to executesoftware embodiments of the present invention. As in FIG. 1, computersystem 1 includes monitor 3 and keyboard 9. Computer system 1 furtherincludes subsystems such as a central processor 102, system memory 104,I/O controller 106, display adapter 108, removable disk 112, fixed disk116, network interface 118, and speaker 120. Removable disk 112 isrepresentative of removable computer readable media like floppies, tape,CD-ROM, removable hard drive, flash memory, and the like. Fixed disk 116is representative of an internal hard drive or the like. Other computersystems suitable for use with the present invention may includeadditional or fewer subsystems. For example, another computer systemcould include more than one processor 102 (i.e., a multi-processorsystem) or memory cache.

Arrows such as 122 represent the system bus architecture of computersystem 1. However, these arrows are illustrative of any interconnectionscheme serving to link the subsystems. For example, display adapter 108may be connected to central processor 102 through a local bus or thesystem may include a memory cache. Computer system 1 shown in FIG. 2 isbut an example of a computer system suitable for use with the presentinvention. Other configurations of subsystems suitable for use with thepresent invention will be readily apparent to one of ordinary skill inthe art. For example, software embodiments of the invention may beimplemented on an IBM compatible computer, workstations from SunMicrosystems, and the like.

Light-directed chemical synthesis combines semiconductor-basedphotolithography and solid phase chemical synthesis. To begin theprocess, linkers modified with photochemically removable protectinggroups are attached to a solid substrate or chip surface. Light isdirected through a photolithographic mask or reticle to specific areasof the synthesis surface, activating those areas for chemical coupling.The first of a series of chemical building blocks (A, C, G, U or T) isincubated with the chip, and chemical coupling occurs at those siteswhich have been illuminated in the preceding step. Next, light isdirected to a different region of the substrate through a new mask, andthe chemical cycle is repeated.

The patterns of light and the order of chemical reagents dictate theidentity of each oligonucleotide probe on the chip surface. Usingcombinatorial synthesis methods, millions of chemical compounds can becreated rapidly in very few process steps.

Oligonucleotide probe arrays contain thousands or millions ofoligonucleotide probes that can be used to recognize longer targetoligonucleotide sequences (for example, from patient samples). Therecognition of sample oligonucleotide by the set of oligonucleotideprobes on the chip takes place through the mechanism of oligonucleotidehybridization. Oligonucleotide hybridization is the simple process inwhich two complementary strands of oligonucleotide join together (Apairs with T and G pairs with C). When an oligonucleotide targethybridizes with an array of oligonucleotide probes, the target will bindto those probes that are complementary to a part of the targetoligonucleotide sequence.

Information about the sequence of the target oligonucleotide may bedetermined according to which probes hybridized with the target. Sucharrays have applications for oligonucleotide probe arrays inoligonucleotide sequence analysis, oligonucleotide sequence checking,mutational analysis, mRNA expression monitoring, and medical diagnosticresearch.

The invention herein provides a technique for synthesizing probe arraysin which the cost of mask manufacturing is reduced. In preferredembodiments of the invention, mask costs are reduced by designing one ora few shift reticles that may be used to synthesize arrays of probes ona substrate. Accordingly, the shift reticle(s) may be used to synthesize"custom" arrays of probes, but the cost of making the mask set for suchcustom probes is greatly reduced on a per chip basis.

FIG. 3A illustrates a probe set that would be desirable for theevaluation of nucleic acid samples expected to contain the sequenceTGACAT. To evaluate a sample to determine if its sequence is, in fact,TGACAT a set of 3-mer probes as shown in FIG. 3A may be synthesized on asubstrate. If a particular sample did have the sequence TGACAT (the"target" sequence), it would be expected to hybridize to each of theprobes ACT, CTG, TGT, and GTA as they are complementary. If, however,there was a variation in the second base position ("G"), lowerhybridization would likely be observed in the ACT probe region as thereis a single base mismatch to the target. Suppose, for example, aparticular sample had the sequence TAACAT. Hybridization would notlikely be observed in the ACT probe region since ACT is not perfectlycomplementary to the sequence TAACAT at any position.

As disclosed in PCT WO95/11995, additional probes may be synthesized todetermine which variation is present at a particular position. Forexample, in addition to the ACT probe, the probes AAT, ATT, and AGT maybe synthesized on the substrate (the interrogation position isunderlined). The strong hybridization of the probe ATT, for example,would indicate that the sample is likely to be TAACAT.

FIG. 3B illustrates probe sets that would be desirable for determiningmutations in samples expected to contain the sequence TGACAT. Eachcolumn contains a set of four probes for determining a nucleotide in thesample corresponding to the interrogation position. As shown, each ofthe four probes in a column differ at the interrogation position. In apreferred embodiment, probes with the same nucleotide at theirrespective interrogation position are placed in a row, thereby formingan A-lane, C-lane, T-lane, and G-lane. The wild-type probes from FIG. 3Aare designated with a "*" within the probe region. Typically, there aremany multiples (e.g., hundreds or thousands) of identical probes withina probe region or cell.

FIG. 4 shows prior art reticles that would be utilized to synthesize theACT, CTG, TGT, and GTA probes of FIG. 3A. Reticles 1 and 6 are foradding the nucleotide A onto the substrate. Reticle 2 is for addingnucleotide C onto the substrate. Reticle 4 is for adding nucleotide Gonto the substrate. Lastly, reticles 3 and 5 are for adding thenucleotide T onto the substrate. Utilizing these reticles, the synthesiscan be viewed as repetitive additions of A, C, T, and then G, withunnecessary addition steps skipped.

In the figures depicting reticles, shaded portions represent openingsthrough the reticle through which light will deprotect areas on thesubstrate. Monomers (e.g., nucleotides) will then be washed over thesubstrate so that the monomers may bind in the deprotected regions.Although in preferred embodiments, the monomer addition regions of thereticles are openings, the monomer addition regions may be closed on thereticles in a similar matter.

FIG. 5 shows the prior art addition of nucleotide monomers to producethe probe set. Reticles 1-6 are utilized to sequentially add monomers toa substrate 202. At the top of FIG. 5, reticle 1 is utilized to add thenucleotide A to the substrate at a location specified by the monomeraddition region of the reticle. Then, reticle 2 is utilized to add thenucleotide C to the substrate as specified by the reticle. The processcontinues through reticle 6 which results in four probes that may beutilized to analyze the target sequence.

Although the process in FIG. 5 can be viewed as repetitive additions ofA, C, T, and G, in some instances, a particular monomer addition stepmay be "skipped." For example, in FIG. 5, the additions of A and C afterthe G addition are skipped in the second cycle of A, C, T, G additions.Thus, six reticles are needed for the synthesis of these probes. In theworst case, an addition of A, C, T, and G would be needed for each ofthe n monomers in the probes. Accordingly, in the worst case, n*4reticles would be needed to synthesize a probe set. In many cases, thisnumber is reduced to some number m where the sequence allows, as in theabove example, where m would be 6 which is better than the worst case of12 (3*4) reticles. As the number of monomers in the probes grows,however, the number of required reticles can become quite large therebyincreasing costs.

The present invention provides techniques for synthesizing probe arraysusing far fewer reticles, which greatly reduces costs. With oneembodiment of the invention, as few as one reticle may be used to make,for example, the exact complement probe set. An additional reticle maybe utilized to make probes with nucleotide variations at aninterrogation position and other reticles may be utilized to fabricatedifferent probe sets on the substrate (e.g., probe sets with differentprobe lengths).

Set of Shift Reticles

In one embodiment, the present invention utilizes a set of shiftreticles to synthesize desired probes on a substrate. The set of shiftreticles includes a single reticle for each monomer that is to be addedto the substrate. Utilizing these reticles, the length and interrogationposition of the probes may be specified at synthesis time, e.g., afterthe reticles have been generated.

FIG. 6 shows a high level flow of a process of generating a shiftreticle set. At step 252, the nucleotides in the perfect complement ofthe target sequence are numbered. Thus, for the target sequence shown inFIG. 3A, the nucleotides may be numbered 1-6. Four 1×6 reticles willsubsequently be formed for synthesizing probes to detect the targetsequence. Each of the four reticles will be utilized to add a differentmonomer (e.g., A, C, G, T) onto the substrate.

At step 254, a reticle for adding the nucleotide A to the substrate iscreated. The reticle is designed with openings corresponding to each Ain the perfect complement to the target sequence. Thus, for the perfectcomplement ACTGTA, the reticle would have openings corresponding tonucleotides 1 and 6. Reticle 1 of FIG. 7 shows a reticle produced inthis manner.

At step 256, a reticle for adding the nucleotide C is created in asimilar manner. The reticle is designed with openings corresponding toeach C in the perfect complement to the target sequence. Therefore, thereticle is designed with an opening corresponding to nucleotide 2 in thetarget. Reticle 2 of FIG. 7 shows a reticle for adding nucleotide C.Steps 258 and 260 create reticles 3 and 4 shown in FIG. 7 in a similarmanner for nucleotides G and T, respectively.

At step 262, a computer file containing the design of the masks isoutput. This file may be utilized by a mask generating system to producethe masks used in synthesis. A system for designing masks is describedin U.S. Pat. No. 5,571,639, which is hereby incorporated by referencefor all purposes.

FIGS. 8A-8C show the addition of monomers using the shift reticles toproduce the probe set. In FIG. 8A, the shift reticles are used toproduce a single "layer" of monomers on a substrate 302. By a "layer" ofmonomers, it is meant that each synthesis cycle of monomer additionsteps uniformly adds monomers to specified locations on the substrate.As shown in FIG. 8A, the specified locations may include the entireactive region of the substrate. However, as will be shown in FIG. 18,the specified locations may include only a subset of locations of theactive region.

Referring still to FIG. 8A, reticle 1 is initially used to add thenucleotide A onto the substrate. Reticle 2 is subsequently utilized toadd the nucleotide C to the substrate, which is followed by the additionof nucleotides G and T utilizing reticles 3 and 4, respectively. Withone synthesis cycle of A, C, G, and then T, a single layer of monomershas been added to the active region of the substrate which is shown asthe four centermost positions on the substrate. The number inparenthesis indicates the reticle being used. The arrow indicates wherethe reticles are aligned with the substrate (i.e., the first possibleopening in each reticle). The reticles will all be shifted one positionafter each synthesis cycle.

In FIG. 8B, the reticles are shifted one position to the left relativeto the substrate as indicated by the arrow in the figure (compare toFIG. 8A). Each reticle is then cycled through to add each of thedifferent monomers onto the substrate at locations specified by thereticles. Again, the reticles add a single layer of monomers to theactive region of the substrate. Although the reticles are shown shiftedor translated to the left, the reticles may, of course, be shifted tothe right or any other direction.

In FIG. 8C, the reticles are again left shifted by one position asindicated by the arrow in the figure. The reticles are cycled throughadding nucleotides A, C, G, and T to the substrate as specified by theopenings in the reticles. After the last reticle is utilized, fourprobes that are perfectly complementary to the target sequence have beensynthesized at the centermost positions of the substrate as shown at thebottom of FIG. 8C. These four probes represent the active region of thesubstrate. The probes shown that are not in the active region of thesubstrate will be called the "edge" of the substrate. These edge probesare typically ignored during analysis or sequencing of a sample.

For simplicity, the monomer addition regions of a reticle have beenshown to add a single monomer onto the subject substrate. In practice,each monomer addition region of a reticle adds hundreds or thousands ofmonomers to the area specified by the opening. Similarly, the reticlestypically synthesize hundreds of rows of probes on the substrate. In apreferred embodiment, the probes are synthesized in multiples of fourrows where the probes in each row differ from the other by a singlenucleotide at an interrogation position.

FIGS. 9A-9B show reticles that may be used to produce multiple probesets for the target sequence shown in FIG. 3. FIG. 9A shows a reticlesimilar to reticle 1 in FIG. 7. However, this reticle would be utilizedto produce two sets of four rows of probes. Likewise, FIG. 9B shows areticle similar to reticle 2 in FIG. 7 that would be utilized to producetwo sets of four rows of probes. The reticles for nucleotides G and Tare not shown but would be similarly produced.

Each of these four shift reticles would be utilized to produce two setsof four rows of probes on the substrate that would be complementary tothe target sequence. Each synthesis cycle in the synthesis produces aset of n-mer complementary probes to this target. Thus, after threecycles through the shift reticles, the substrate contains a set of 3-mercomplementary probes. The length of complementary probes may be selectedat synthesis time by the number of synthesis cycles of monomer additionsteps that are used, where a "synthesis cycle" is defined as cyclingthrough each of the monomers to be added to the substrate. One synthesiscycle results in adding a layer of monomers to specified locations onthe substrate, typically the active region of the substrate.

FIG. 10 shows the transformation of a linear reticle into a rectangularreticle. Although the shift reticles have been shown as long linearreticles, the linear reticles may be transformed into a rectangularshift reticle. A linear reticle 302 includes sixteen active cells andthree extra cells. The extra cells are the cells added to the reticlefor shifting relative to the substrate. Typically the number of cells inthe linear shift reticle will be substantially greater but the simplereticle is shown for illustration purposes.

When transforming the linear reticle into a rectangular reticle, thesixteen active cells are placed in a rectangular region with theappropriate extra cells at the edge. Thus, a rectangular reticle 304 wasformed by placing cells 1-16 into a square region of the reticle. Eachrow of the rectangular reticle ended with the same number of extra cellsas was in the linear reticle, these extra cells continuing sequentiallyafter the active cells. The resulting rectangular reticle is a shiftreticle that forms rows of probes for a target sequence.

In order to add differing monomers at an interrogation position,reticles such as those shown in FIGS. 11A and 12A are utilized. FIG. 11Ashows a reticle that is used to couple interrogation position monomerson the substrate. The reticle includes multiple rows of openings thatare utilized to add a monomer to the probes. These rows areperpendicular to the reticles shown in FIGS. 9A and 9B.

The reticle in FIG. 11A is first utilized to add a monomer likenucleotide A to a row of probes (i.e., the A-lane). The reticle is thenshifted downwards to the next row of probes and a different monomer likenucleotide C is then added to this row of probes (i.e., the C-lane).This process is continued until a different monomer is added to theinterrogation position for each row in a set of probes. For a deletion,a monomer is not added to a row of probes.

Thus, in order to produce 5-mer probes with an interrogation position atthe third position in the probes, one performs two synthesis cycles ofmonomer addition steps with the shift reticles to produce 2-mer probesin the active region of the substrate. Then the interrogation positionreticle is utilized to add a different monomer to each row of probes byshifting the mask in a direction perpendicular to the direction that theshift reticles are shifted. Except in the case of deletions, theinterrogation position reticle also adds a layer of monomers in theactive region of the substrate with one synthesis cycle. Then, aftershifting the shift reticles two positions (the extra position accountsfor the synthesis cycle utilized by the interrogation position reticle),the shift reticles are utilized to add the last two monomers to theprobes by performing two synthesis cycles of monomer addition steps.

FIG. 11B shows probes on a chip that vary at an interrogation position.The top half of the chip includes 4-mer probes with the interrogationposition at the third nucleotide in the probe (the interrogationnucleotide is circled for easy identification). As shown, there are4-mer probes in the active region of this half of the chip. The bottomhalf of the chip includes 3-mer probes in the active region that weresynthesized using the same shift reticles and the interrogation positionreticle at the same time with the use of one additional reticle whichallows different length probes to be synthesized on the chip at the sametime. This reticle will be described in more detail in reference to FIG.13A.

The shift reticles of the present invention are target structurespecific but not sequence specific. For example, the shift reticles maybe utilized to synthesize probes complementary to the sense oranti-sense strands of DNA. Additionally, shift reticles that produceprobes complementary to TGACAT may also be used to produce probescomplementary to AGTCTA by switching the A and T nucleotides utilizedwith the shift reticles. Accordingly, at synthesis time, one may specifycharacteristics of the probes by selecting the order of shift reticlesin a synthesis cycle, the monomers in a synthesis cycle, the monomersassociated with each of the shift reticles, and the interrogationposition.

FIG. 11C is an image of a chip that was produced in the manner describedabove. The chip has 20-mer probes with an interrogation position at acentral position. The chip is for sequencing a 1000 base-pair sequenceof HIV. It should be understood that the examples described above aresimplified to aid the readers understanding of the invention. The chipimages show actual chips and are therefore more complex, but theynevertheless utilize the principles of the present invention extended toa larger scale.

The probes in the edge regions of the substrate will still bind to thelabeled target with varying hybridization intensities as shown on theright side of the chip in FIG. 11C. However, it is difficult for one tovisually identify where the active region of the chip begins or ends. Ina preferred embodiment, an interrogation position reticle as shown inFIG. 12A is utilized. This reticle has openings that only correspond toor overly the active region of the substrate. Because the openings willnot be above the edge regions of the substrate, the probes at the edgeregions of the substrate will not receive the interrogation positionnucleotides. In this manner, the probes in each column in the edgeregions will be identical and therefore, the edge region will appear asstripes in the image of the chip so the start of the active region maybe more easily identified. This is shown on the left side of the chip inFIG. 11C.

FIG. 12B shows probes on a chip that vary at an interrogation positionbut were synthesized utilizing the reticle shown in FIG. 12A. The tophalf of the chip includes 4-mer probes with the interrogation positionat the third nucleotide in the probe. As shown, there are 4-mer probesin the active region lane of this half of the chip. The circledinterrogation nucleotides were only added in the active region. Thus,the probes in each column outside the active region (edges) areidentical. As they are identical, the resulting stripes may be utilizedto identify the edges of the chip after hybridization and scanning. Thebottom half of the chip includes 3-mer probes that were synthesizedusing the reticle shown in FIG. 13A.

A chip synthesized as described above is shown in FIG. 12C where theedge regions may be easily identified by the stripes. The chip has20-mer probes with an interrogation position at a central position. Thechip is for sequencing a 2,500 base-pair sequence of HIV.

Utilizing these shift reticles and the interrogation position reticle,any length probe with any substitution position may be synthesized for atarget sequence limited only by the size of the reticles. Typically, thesize of the reticles is equal to the size of the target along a row ofthe substrate plus the desired length of the synthesized probes minusone. For example, if there are 100 columns of cells on the chip and thetarget sequence is equal to or longer than 100 monomers, the reticlesmay be 111 cells (or possible monomer addition regions) wide for 12-merprobes (i.e., 100+12-1).

With the present invention, five reticles may be utilized to sequenceany length probe with any interrogation position for the targetsequence. Furthermore, the length of the probes and the interrogationposition need not be determined before synthesis. After the reticles areproduced, the specific probes that are produced on the substrate may bedetermined at synthesis time by indicating the number of cycles ofmonomer addition steps and the cycle where the interrogation positionreticle will be utilized.

A reticle as shown in FIG. 13A may be utilized to generate probes ofvarying length with the lengths being determined at synthesis time ifdesired. The reticle includes an opening which will allow light tostrike a set of probes. The reticle may be utilized in conjunction withthe shift reticles so that only the top half of the chip is deprotected.For example, the reticle may be utilized to add a first layer ofnucleotides only to the top half of the chip. After the first layer ofnucleotides has been added, synthesis may continue but now by addingnucleotides to the whole chip. It is in this manner that the 4-mer and3-mer probes of FIGS. 11B and 12B were produced.

Alternatively, the reticle of FIG. 13A may be utilized to stopsynthesis. After probes have been synthesized in a region, the regionspecified by the reticle is deprotected and a capping reagent may beadded to the substrate so that subsequent exposure to light will notdeprotect the probes in this region. The capping region may be DMT orany other known capping reagent. Utilizing this reticle, one region ofthe substrate may contain probes that are of a different length thananother area of the substrate. For example, the substrate shown in FIG.13B includes 8-mer probes and 12-mer probes. The 8-mer probes were firstformed on the whole chip and then the reticle of FIG. 13A was utilizedto cap the probes on the top half so that subsequent exposure to lightwould not result in the addition of subsequent monomers. The region forthe 12-mer probes was not capped so the subsequent addition of monomersresulted in 12-mer probes.

Additionally, the reticle shown in FIG. 13A may be utilized tosynthesize probes with nucleotide deletions. For example, by utilizingthe reticle to skip a cycle of nucleotide additions, probes withdeletions may be synthesized.

FIG. 14A is a mask including multiple reticles. A mask 500 includesshift reticles 502, 504, 506, and 508, one for each nucleotide monomer.The mask also includes an interrogation position reticle 510 which addsmonomers at an interrogation position over the active region of thesubstrate. Additionally, the mask includes a reticle 508 which isutilized to deprotect the entire surface of the substrate in order tocap the probes after synthesis. Although FIG. 14A shows a mask thatincludes multiple reticles, the present invention may be advantageouslyutilized in those systems where each mask includes a single reticle.

FIG. 14B shows the layout of a reticle in one embodiment. The majorityof the reticle includes rows with monomer addition regions for repeatinggroups of A, C, G, and T lanes of the substrate. The monomer additionregions for each group of lanes are typically the same as shown in FIGS.9A and 9B. Each group of rows corresponding to the A, C, G, and T lanesmay differ in order to synthesize probes complementary to differentsections of the target. For example, one group may be for synthesizingprobes for identifying nucleotides at positions 500-599 while the nextgroup is for synthesizing probes for identifying nucleotides atpositions 600-699.

The top and bottom rows of the reticle in FIG. 14B are for producingprobes complementary to a control oligonucleotide sequence (i.e.,control probe lanes). The control sequence is a known sequence that isadded to the target to allow easier identification and/or alignment ofthe active region of the chip after scanning.

FIG. 14C shows reticles for synthesizing probes on two chipssimultaneously. As shown, there are identical A reticles, C reticles, Greticles, T reticles, and interrogation position reticles for each chip(denoted chip 1 and chip 2). These reticles reside on the same piece ofglass so that two identical chips may be produced simultaneously. Thus,if the synthesis cycle begins with A, the two A reticles would beutilized. The glass would then be shifted so that the next nucleotidereticle is over the chips to add the next nucleotide in the synthesiscycle, and so forth. At the next synthesis cycle, the reticles would bepositioned over the chip at a position shifted horizontally relative tothe chip. Accordingly, the nucleotide reticles are wider than the chips.

The interrogation position reticles may be utilized to synthesizenucleotides at an interrogation position in the probes on the chips.During the synthesis cycle which is designated to add the interrogationposition nucleotides, the glass is shifted vertically relative to thechip. One should understand that although the nucleotide reticles aredescribed as being shifted horizontally and the interrogation positionreticles as being shifted vertically, the reticles may be shifted anydirection. Also, the reticles for chip 1 and chip 2 need not beidentical, nor limited to two chips. Accordingly, multiple differentchips may be synthesized with the present invention simultaneously.

FIG. 14D shows a mask for synthesizing varying length probes on twochips simultaneously. Ways have been described for utilizing reticlesfor selecting regions of the chip in order to synthesize probes ofvarying length (see, e.g., FIGS. 13A and 13B). Another way of achievingthis objective is illustrated with the mask in FIG. 14D.

The mask includes reticles similar to the reticles described in FIG.14C, and in fact, the reticles in the left bottom of the mask areidentical to these reticles. The underlying chip has five groups of A,C, G, and T lanes of the chip. As shown, the reticles in the left bottomof the mask have rows of monomer addition regions that correspond toeach of the five groups of A, C, G, and T lanes. Each group of A, C, G,and T lanes are identical, however, not all of the reticles have thesame number of groups. There are other reticles with one, two, three,and four groups of A, C, G, and T lanes.

In order to synthesize chips with varying length probes, one selects thereticles that will add monomers at desired regions on the chip. Forexample, if one desires to synthesize 3, 5, 7, 9, and 11-mer probes ontwo chips simultaneously with interrogation positions at the center ofthe probes, one could first use the reticles with the single group of A,C, G, and T lanes for one synthesis cycle. This would couple monomers onthe top portion of the chip.

Next, one could use the reticles with the two groups of A, C, G, and Tlanes for one synthesis cycle. This would synthesize a top region withtwo layers of monomers (i.e., 2-mer probes) and an adjacent region withone layer of monomers. This process may be repeated utilizing thereticles with three, four and five groups of A, C, G, and T lanes untilthere are regions on the chip with five, four, three, two and one layermonomers (from top to bottom of the chip).

The interrogation position reticle at the lower middle of the mask maythen be utilized to add interrogation position nucleotides to all of theprobes on the chip. After the interrogation position reticle has beenutilized, the previous process of adding nucleotides may be reversed.After synthesis, open chip reticles may be utilized to cap the probesthereby generating two chips with 3, 5, 7, 9, and 11-mer probes withinterrogation positions at the center of the probes. The layout of oneof these chips is shown in FIG. 14E.

FIG. 15A shows a layout of a chip in another embodiment which istypically utilized for genotyping or gene expression applications. Asshown in FIG. 15A, a chip 550 has perfect complement lanes 552, mutationlanes 554 and control lanes 556. The perfect complement lane has probesthat are perfectly complementary to the target sequence. The mutationlane has probes that are complementary to the target sequence except fora mutation position. The mutation lanes are utilized to check thevalidity of the data. Thus, hybridization intensities in the perfectcomplement lane are compared to the hybridization intensities in themutation lane.

FIG. 15B shows a shift reticle for coupling a particular monomer on achip in pairs of rows. The reticle may be utilized to add nucleotides toboth the perfect complement and mutation rows. Typically, there will befour reticles, one for each nucleotide (see, e.g., FIG. 7), that areused in each synthesis cycle. However, only one reticle is shown.

FIG. 15C shows a shift reticle for coupling a particular monomer on achip in a single lane. In order to produce probes in the mutation lanethat differ from the probes in the perfect complement, a shift reticleas shown in FIG. 15C may be utilized. As before, there will typically befour reticles, one for adding each of the nucleotides, but one is shownfor simplicity. In one synthesis cycle, these shift reticles may beutilized to add nucleotides that are perfectly complementary to thetarget sequence in the perfect complement lane as was done with thereticle shown in FIG. 15B.

With the invention, the same shift reticle shown in FIG. 15C may beutilized to add mutation nucleotides to the probes in the mutationlanes. The shift reticles are shifted vertically so that the monomeraddition regions overly the mutation lanes. In order to add mutationnucleotides to the probes in the mutation lanes, one may change theorder of the nucleotide addition steps in the synthesis cycle. Forexample, if the nucleotides A, C, G, and then T are added in a synthesiscycle, one can instead add T, G, C, and then A, which is reverse order.Thus, each probe in the mutation lanes will have a mutation nucleotideadded.

Alternatively, one may keep the order of the nucleotide addition stepsbut switch the order of the shift reticles that are utilized. As shouldbe apparent, this has the same effect of adding a mutation nucleotide tothe probes in the mutation lanes.

FIG. 15D shows a shift reticle for forming control lanes that includecontrol probes. The control probes may be perfectly complementary to aknown oligonucleotide that is hybridized with the chip in order to aidin analyzing the scanning results. Again, one shift reticle is shown butthere will typically be one for each monomer.

The above embodiment provides shift reticles which may be utilized toform probes of varying lengths which are complementary to the targetsequence. These shift reticles may be utilized with one or more masks inorder to produce probes with interrogation position nucleotides orprobes of varying length on the same substrate as described. The costfor producing probes on a substrate are reduced because the number ofreticles may be greatly reduced (e.g., down to five reticles or less).Flexibility is increased as one may specify characteristics of theprobes at synthesis time.

Single Shift Reticle

In another embodiment, the present invention provides a single shiftreticle that may be utilized to synthesize probes complementary to thetarget sequence. FIG. 16 shows a high level flow of a process ofgenerating reticles according to this embodiment of the invention. Atstep 602, the nucleotides in the perfect complement of the targetsequence are numbered. If the target sequence is TGACAT as shown in FIG.3A, the perfect complement will be ACTGTA. Thus, the nucleotides in theperfect complement are numbered 1-6 with the first A being 1, C being 2,the first T being 3, and so forth.

A single shift reticle is then produced according to steps 604-610. Itshould be noted that these steps do not need to be performed in anyspecific order and in fact, they may be performed in parallel.Furthermore, each equation is not specific to the nucleotide shown.However, the steps will be described as being performed sequentially foreach nucleotide A, C, G, and T for ease of illustration.

At step 604, openings are created in the single reticle for each A inthe perfect complement by the equation n*(i-1)+1, where n is equal tothe number of different types of monomers (e.g., nucleotides) and i isequal to a position of the monomer in the perfect complement (or desiredprobe). As the nucleotide A is at base positions 1 and 6 in the perfectcomplement, openings will be created in the single reticle at position 1and 21 because n is equal to 4 for the four nucleotides A, C, G, and T,and i is equal to 1 for the first A and 6 for the second A. FIG. 17Ashows the resulting single reticle.

At step 606, openings are created in the single reticle for each C inthe perfect complement by the equation n*(i-1)+2, where n is equal tothe number of different types of monomers and i is equal to a positionof the monomer in the perfect complement. As the nucleotide C is at baseposition 2 in the perfect complement, an opening will be created in thesingle reticle at position 6 because n is equal to 4 and i is equal to2. FIG. 17B shows the single reticle with openings for both A and C.

Openings are created in the single reticle for each G in the perfectcomplement by the equation n*(i-1)+3 at step 608. As the nucleotide G isat base position 4 in the perfect complement, an opening will be createdin the single reticle at position 15 because n is equal to 4 and i isequal to 2. FIG. 17C shows the single reticle with openings for A, C andG.

At step 610, openings are created in the single reticle for each T inthe perfect complement by the equation n*(i-1)+4. As the nucleotide T isat base positions 3 and 5 in the perfect complement, openings will becreated in the single reticle at positions 12 and 20 because n is equalto 4 and i is equal to 2. FIG. 17D shows the single reticle withopenings for A, C, G, and T.

At step 612, a mask file for generating a mask including the singlereticle is output. This mask file is typically utilized by a computeroperated system to generate the mask.

FIG. 18 shows a single shift reticle that produces the probe set of FIG.3A and the addition of monomers using this reticle. Reticle 652 isproduced according to the process described in reference to FIG. 16. Asshown, the reticle includes six cycles of A, C, G, and T (denoted 1-6above the reticle). Each cycle includes a single opening at variouspositions as shown.

Initially, the mask is utilized to add the nucleotide A to the substrateat the regions specified by the mask. With each subsequent synthesisstep, the reticle is shifted by one position or cell with each step,resulting in four shifts for each synthesis cycle of nucleotides. Thisprocess is shown in a table 654 underneath the reticle with thenucleotide addition steps sequentially listed on the left side of thetable. The dashed line in the table represents the rightmost border ofthe active region of the substrate. In other words, nucleotides to theright of the dashed line would not be coupled to the substrate.

The table is typically not utilized during synthesis but is shown to aidin understanding how the probes on the substrate in this embodiment areformed. Each column in the table represents a probe on the substrate.However, as the table grows downward as monomers are added, the firstnucleotide from the top in each column is nearer the substrate.

A substrate 656 results with the desired 3-mer probes indicated by thefour arrows underneath the substrate. The desired probes are formed by auniform addition of nucleotides at these specified regions because eachcycle adds one nucleotide to each desired probe. Accordingly, aninterrogation position reticle may be utilized that is similar to theones shown in FIG. 11A and 12A in order to add interrogation positionnucleotides. After the interrogation position nucleotides are added, asynthesis cycle of the single shift reticle is then skipped so thereticle is shifted four positions or cells (e.g., to the left in FIG.18).

As shown, there are a number of "junk" probes surrounding the desiredprobes. Typically these probes will be ignored during sequencing of thetarget. For simplicity, the single reticle has been shown as a linearreticle. However, a reticle may be utilized for producing two sets offour rows of probes as shown in FIG. 19. More rows of probes may begenerated by an extension of the principles of the invention.

Although the single shift reticle has been shown as a long linearreticle, the linear reticle may be transformed into a rectangular shiftreticle as shown in FIG. 10. As the single shift reticle is shifted witheach monomer addition step, the number of extra cells at the end of eachrow in the resulting rectangular reticle may be substantially higher.

This embodiment of the present invention allows probes perfectlycomplementary to the target sequence to be synthesized on the substratewith a single shift reticle. Additional reticles may be utilized tosynthesize probes with interrogation position nucleotides or probes ofvarying lengths as described above. By reducing the number of reticlesneeded down to possibly one, this embodiment greatly reduces the cost ofgenerating masks for probe array synthesis. Additionally, flexibility isincreased because characteristics of the desired probes may be specifiedat synthesis time.

Other Shift Reticle Embodiments

In another embodiment, the present invention provides shift reticlesthat may be utilized to synthesize probes for detecting mutations,deletions, and the like. These shift reticles are not target sequencestructure specific so the target sequence may be specified at synthesistime. In other words, a set of "generic" shift reticles may be utilizedto synthesize probes for analyzing any target sequence. Additionally,these probes may be generated with very few reticles.

FIGS. 20A-20D show shift reticles for synthesizing probes of includingmultiple monomers for detecting mutations and a deletion. In order toillustrate how the shift reticles work it may be beneficial to discussan example. Suppose it is desired to synthesize probes that would detecta mutation or deletion at the middle (or 8th) position in a 15-mertarget. It should be understood that typically target sequences are muchlonger but this example will be utilized to illustrate the invention.

If the target sequence is TACCGTGAAGCTACG (SEQ ID NO:1) then it would bedesirable to synthesize the following probes: ATGGCACTTCGATGC, (SEQ IDNO:2) ATGGCACGTCGATGC, (SEQ ID NO:3) ATGGCACCTCGATGC, (SEQ ID NO:4)ATGGCACATCGATGC, (SEQ ID NO:5) and ATGGCACTCGATGC (SEQ ID No:6). Theinterrogation position nucleotides are underlined which illustrates thatthe first probe is the perfect complement to the target sequence. Thenext three probes have a mutation at the interrogation position and thelast probe has a deletion at the interrogation position.

Four shift reticles (or less) may be utilized to synthesize theseprobes. The shift reticle in FIG. 20A is utilized for couplingnon-interrogation position monomers to the substrate. Nucleotideaddition steps are cycled through that correspond to the complement ofthe target sequence. As indicated by the nucleotides above the shiftreticle, first A is added, then T, then G, and so forth. After eachmonomer addition step, the shift reticle is shifted one position to theleft. As the shift reticle is shown as being five rows high, fiveidentical probes will be generated up to the 8th monomer addition step.

At the 8th monomer addition step, which corresponds to T in the perfectcomplement, only one monomer addition region overlies the probes.Accordingly, T will be only added to one of the probes, which is the topprobe in the FIG. 20A.

FIG. 20B shows a reticle that has a single monomer addition region. Thereticle may be utilized to add a G at the 8th position of the secondprobe from the top. Similarly, the reticles in FIG. 20C and 20D may beutilized to add a C and A at the 8th position of the probescorresponding to the one monomer addition region of the reticles. Theprobe at the bottom does not have a monomer added at the 8th position sothat a deletion at this position may be detected. Although FIGS. 20B-20Dshow three shift reticles, a single shift reticle may be utilized thatis shifted vertically.

After the interrogation position nucleotides are added, the shiftreticle of FIG. 20A is utilized to add the rest of the nucleotides tothe probes. FIG. 20E shows a chip including these probes. A chip 800includes a perfectly complementary probe, interrogation position probes,and a probe for a deletion (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, and SEQ ID NO:6).

The shift reticle described above may be modified to produce probes ofvarying lengths. FIG. 21A shows a shift reticle for synthesizing relatedprobes of varying lengths on a substrate. The shift reticle has monomeraddition regions that vary in width to produce 9, 11, 13 15, 17, 19, and21 -mer probes. As the shift reticle is shifted along the directionindicated by the arrow in FIG. 21A, monomers are added to the substrate.

The varying widths of the monomer addition regions may be designed toresult in varying length probes that are centered around the sameposition of a target sequence. For example, as shown in FIG. 21A, eachmonomer addition step is labeled from 1-21. At step 1, only one regionwill have a monomer added. Each subsequent step adds a monomer to thisregion and the region above it. In this way, the stair-step design ofthe shift reticle allows varying length probes to be synthesized on thesubstrate. By utilizing a stair-step design at each end of the shiftreticle, the probes vary by two monomers instead of one which would beachieved with a single stair-step.

FIG. 21B shows an example of the layout of the probes that may besynthesized on the substrate. A chip 850 includes regions that include9, 11, 13, 15, 17, 19, and 21-mer probes. Although it has only beendescribed that there is one row for each length probe, there may bemultiple rows for each length probe. For example, there may be four rowsfor each length probe, one for nucleotide at an interrogation position.At the interrogation position, an interrogation position reticle as wasdescribed in reference to FIGS. 11A and 12A may be utilized to add theinterrogation position nucleotides instead of utilizing the shiftreticle. An advantage of the invention is that the sequence of theprobes, the length of the probes, and interrogation position may beselected at synthesis time.

This embodiment of the present invention allows probes of varyinglengths and that are centered around a position in the target sequenceto be synthesized. The shift reticle may also be utilized with aninterrogation position reticle to produce varying length probes thatdetect mutations.

These embodiments of the invention have the significant advantage thatthe shift reticles are not target sequence structure specific.Accordingly, the sequence of the target may be specified at synthesistime and a "generic" set of shift reticles utilized to synthesize probesfor analyzing the target sequence. As with the other embodiments of theinvention, the number of reticles needed is significantly reduced whichlowers the cost of producing the chips. Also, flexibility is increasedbecause characteristics (e.g., interrogation position) of the desiredprobes may be specified at synthesis time.

Speckle Masks

Some embodiments of the present invention utilize speckle masks. A"speckle mask" is a set of reticles that when taken together have anopening at each location, thus they, in effect, can be said to form afull open mask. FIG. 22 is a simple example of speckle masks. As shown,the three reticles have a single monomer addition opening at a differentlocation. When the openings are added together, a full open mask isgenerated. This is a property of speckle masks.

Another example of a speckle mask is the set of reticles (or masks)shown in FIG. 7. These masks, together include one and only one openingfor each monomer addition region in the reticles. FIG. 23 shows how theopenings of the shift reticles add up to form a full open mask.

A fundamental property of speckle masks is that if all the reticles areused in a synthesis cycle, exactly one monomer is added to each of theprobes in the active region of the substrate. This property is used togreat effect in allowing construction of probes of any length andinterrogation position at synthesis time.

Another application of speckle masks is to generate a number of distinctchips from a single speckle set. Take a grid and construct a speckle setby assigning random numbers from 1-4 (or whatever the number of monomershappens to be) in each cell. The number indicates which reticle willhave an opening at that location. If all four reticles are cycledthrough with some permutation of A, C, G, and T in a synthesis cycle, aset of "random" nucleotides are added to each probe on the substrate. Ifsome arbitrary (x and y) offset is utilized in each step, very littlecorrelation between the nucleotides added to each probe is expected. Foreach distinct set of offsets, radically different sets of probes may begenerated. Thus, "random" chips with probes of uniform length(neglecting probes on the edges of the chip) may be generated.

A further application of a speckle set is to generate a chosen set ofuniform length probes. A shift mask may be generated that produces aspecific set of probes by picking a sequence containing that set ofprobes, and generating a shift mask to that sequence. However, thesequence containing some set of probes will in general be very muchlonger than the total number of probes. Since a shift mask contains anumber of cells approximating the total length of the sequence, this maybe an inefficient way of generating some sets of probes.

A shift mask uses one-dimensional offsets to generate the probes. A wayof looking at this is that each probe must be encoded on the mask in astrip 1×n, where n is the length of the probe. The strips are packedonto the mask set to produce the set of probes. Any pair of strips mayonly interact in O(n) ways, corresponding to the number of ways therectangles may overlap.

A better method of packing probes onto a speckle set is to usetwo-dimensional offsets. With 2-dimensional offsets, probes are encodedon the mask in "speckles" some arrangement of n cells (where n is thelength of the probe). In general, there are O(n²) ways for two specklesto interact. This suggests that two-dimensional offsets may be used topack probes efficiently in a speckle set. However, this problem appearscomputationally very difficult, given the degrees of freedom to chooseoffsets, base permutation used at each synthesis cycle, and probelocation. Some form of simulated annealing could be used to chooselocations, given the chosen set of offsets and base permutation.

FIG. 24 shows the packing of speckle masks. Two sequence ACTGT and ATCTGmay be packed by taking advantage of the common subsequence CTG. Thepacking of the speckle masks involves both an x and y offset as shown.

Several possible generalizations of speckle sets exist. One may use anumber of masks greater than the set of bases used to increase thenumber of degrees of freedom. One may also generate sets of masks thatadd up to several open masks (each cell is open exactly k times, whenthe full set of masks is taken together). Additionally, one may generatesets of masks that have many different subsets that add up to an openmask.

Post Chip Synthesis

In the embodiments described above, the reticles were designed asrectangular grids. The rectangular grids are utilized as it lends itselfwell to switch matrix representation. Switch matrices provide anexcellent generalization of combinatorial masks, but they generallyrequire that the chips include an array of rectangular cells, where allof the cells are the same size. These chips may include wasted space asthe blank lanes (lanes including no probes) are the same size as laneswhich include probes.

With post chip synthesis, each set of related probes (e.g., probesvarying by a single base at an interrogation position) are treated as acharacter in a text document. A set of related probes will be referredto as an "analysis region." Just as characters are not restricted torectangular grids in modern printers, analysis regions are also notlimited but instead may be scaled, rotated, stretched or manipulated.Accordingly, the analysis regions may be input as a sequential list.

FIG. 25 shows the layout of a chip in one post chip synthesisembodiment. An analysis region 900 includes four cells denoted G, T, A,and C to indicate the nucleotide at the interrogation position of theprobes in each cell. As shown, the analysis regions are placed in acircular pattern. Although only one ring of analysis regions is shown,more rings may be generated around a center. This pattern of analysisregions may be extended to resemble data that is stored on hard drives,including sectors and tracks, for reading by a computer controlleddevice.

FIG. 26 shows the layout of another chip utilizing post chip synthesis.In this embodiment, the analysis regions are synthesized in a spiralpattern, similar to a phonographic record. Additionally, the masks thatsynthesized the probes on the chips did not add the nucleotides in arectangular cell. Instead, the probes were synthesized on the chip in anoutline of the interrogation nucleotide letter: A, C, G, or T. Asexpected, the probes that best hybridize with the sample sequence showngenerate the highest intensity, which will form the brightest outline ofone of these characters. In other words, a person may be able to justread the bases right off the scan image.

Alternatively, a computer system may utilize optical characterrecognition techniques to read the characters indicative of theinterrogation base from the scan image. This process may be furtheradded by the spiral placement of the analysis regions.

With post chip synthesis, analysis regions may be placed in differingorientations, spirals, or with variable spacing between the analysisregions. Flexibility in laying out the chip is provided which may proveto be very beneficial in many applications.

Edge Minimization

In order to maximize the utilization of the active region of thesubstrate, it may be beneficial to pack groups of A, C, G, and T-lanestogether with no blank lanes in between. FIG. 27A shows an active region1000 of a substrate that includes multiple groups of A, C, G, andT-lanes 1002. As shown, there are no blank lanes separating each groupof lanes 1002. Although this may appear to be the best utilization ofthe real estate of the active region, the synthesis of the probes in theT-lane of one group may adversely affect the synthesis of probes in anadjacent A-lane of another group.

In order to show how the synthesis of one group may affect another, FIG.27B shows a subregion of eight cells 1004 from FIG. 27A. As shown, fourof the cells 1050, 1052, 1054, and 1056 are from a first group of A, C,G, and T-lanes and four cells 1058, 1060, 1062, and 1064 are from anadjacent group. Within each cell are 5-mer probes with an interrogationposition at the third position (underlined). In practice, the probes aretypically longer than 5-mers but shorter probes are shown to benefit thereader.

When the cells on the substrate are tightly packed, data from cells(e.g., cells 1056 and 1058) that are adjacent to another group of cellsis not as accurate. The reason for this is that the probe AGTAT fromcell 1056 and the probe GCAAA from cell 1058 only have one base incommon, the fourth base in both probes is an A. Therefore, duringsynthesis, many of the masks will have an opening for only one of thesecells, which creates an "edge" on the mask between the two cells.Accordingly, it can be said that there are four edges on the reticlesutilized to generate the probes in cells 1058 and 1060.

In stark contrast, the probe AGGAT from cell 1054 and the probe AGTATfrom cell 1056 have four bases in common. As these two probes are fromthe same group of probes, only the interrogation position bases differ.Thus, it can be said that there is only one edge on the reticlesutilized to generate the probes in cells 1056 and 1058. The significanceof the number of edges is described below.

Light tends to diffuse somewhat around an edge of a reticle so the moreedges that are present between two cells, the more it is that the cellswill have incorrect probes near the edge. As described above, there werefour edges between cells 1056 and 1058, whereas there was only one edgebetween cells 1054 and 1056. Accordingly, the data from probes near theborder between cells 1056 and 1058 will likely be less accurate.Although synthesizing a blank lane between the groups of A, C, G, andT-lanes reduces this "edge effect," the reduction is only approximatelyone half since there will still be edges for the generation of the blanklane.

The present invention reduces the number of edges by utilizing shiftreticles that synthesize non-interrogation position bases in an areathat is wider that the area in which interrogation position bases aresynthesized. For example, the shift reticles shown in FIGS. 9A and 9Bhave monomer addition regions that are four cells wide. The monomeraddition regions may be widened to five cells wide so thatnon-interrogation position bases are synthesized in an area on the chipthat is five cells wide. An interrogation shift reticle, such as shownin FIGS. 11A and 12A, may then be utilized to synthesize interrogationposition reticles in an area that is narrower (e.g., four cells wide)than the area occupied by the non-interrogation position bases.

In order to more clearly see how the invention provides a reduction inedges, FIG. 27C shows a subregion of FIG. 27B that may be synthesizedwith reduced edges. A subregion 1004' includes eight cells 1050, 1052,1054, 1056, 1058, 1060, 1062, and 1064 that are the same as those inFIG. 27B that have the same reference numerals. However, in subregion1004, the non-interrogation position bases are synthesized five cellswide. Accordingly, there are half cells 1070 and 1072 surrounding eachof the multiple groups of A, C, G, and T-lanes 1002.

Half cells 1070 include the same bases as the probes in cells 1050 and1056 except for a single additional base, the interrogation base.Therefore, there is only one edge difference between half cells 1070 andthe full cells they border. As described above, there is only a one edgedifference between, e.g., cells 1054 and 1056. Therefore, each of cells1050, 1052, 1054, and 1056 have the same number of edges at theirborders so they should provide more accurate data.

Although in preferred embodiments, the non-interrogation position basesare synthesized in an area five cells wide, this exact size is notrequired. Edges may be reduced when the non-interrogation position basesare synthesized in an area that is wider than the area in which theinterrogation position bases are synthesized. It may seem that havingunused space between groups of lanes would waste real estate in theactive area on the chip. However, it has been found that because thedata is more accurate, the feature sizes may be reduced more so that thedensity of cells may actually be increased.

Probe Optimization

In some instances, it may be beneficial to synthesize various probesthat interrogate a specific base position in a target. For example, onemay only be interested in specific point mutations in a gene. In orderto fully interrogate the specific base, it would be beneficial to havemany different probes (e.g., length and/or interrogation position in theprobe) that interrogate the position.

An embodiment of the invention allows one to synthesize different probesfor interrogating a specific base position. Conceptually, the inventioncombines the non-interrogation base reticles with the interrogationposition reticle. FIG. 28 shows shift reticles that produce equal lengthprobes with different interrogation positions. It should be understoodthat in this instance, "interrogation position" means the position inthe probe that interrogates a position in the target. The "interrogationposition" may also refer to the position in the target that is beinginterrogated.

Assume a target was AGCGATANCTGCGTA, (SEQ ID NO:7) where the underlinedN designates an unknown base at an interrogation position. The shiftreticles of FIG. 28 may be created as described in reference to FIG. 7.The bases shown on top of the shift reticles are merely a reference tothe corresponding base in the target and an asterisk 1102 indicates theinterrogation position. The cells at this location in the reticles willbe formed similar to the interrogation position reticles of FIG. 11A or12A. As shown, at the interrogation position, a different monomeraddition region is generated for each reticle. For example, Reticle 1(for A) has a monomer addition region in the A-lane, Reticle 2 (for C)has a monomer addition region in the C-lane, and so forth.

When the shift reticles of FIG. 28 are utilized, there is no need for aninterrogation position reticle. After eight cycles through the additionof A, C, G, and T with the shift reticles, 8-mer probes would besynthesized that have all the possible interrogation positions in theprobes.

FIG. 29 illustrates an example of the position of the 8-mer probes. Achip 1150 has eight different sets of four probes in its active region.The number below (1-8) indicates the position of the interrogationposition in the probes if the shift reticles of FIG. 28 are shifted tothe left after each monomer addition step. A "1" indicates thatinterrogation position is nearer the chip in the probes, whereas an "8"indicates that the interrogation position is farther from the chip inthe probes.

By utilizing the shift masks in FIG. 28, one may synthesize probes of aspecific length with every possible interrogation position. Although8-mers have been described as an example, the invention is not limitedto any specific probe length. Additionally, probes may be furtheroptimized by having probes of different lengths synthesized on the chipat the same time as follows.

FIG. 30 shows a shift reticle 1200 for producing probes with differentlengths and interrogation positions. The top half of the shift reticle1202 is the same as Reticle 1 of FIG. 28. Accordingly, it may beutilized to form 8-mer probes with different interrogation positions.The bottom half of the shift reticle 1204 is similar to the top halfexcept that it has two "blank" positions 1206. These blank positionswill not have any monomer addition regions in any of the shift reticles.Because there are two base positions that are blank, the bottom half ofthe shift reticle does not cover as much of the target as the top half.For simplicity, only one shift reticle is shown but it should be readilyunderstood that for nucleic acid applications, there will be three othershift reticles for the other three bases.

FIG. 31. illustrates the probes that may be produced by shift reticlesaccording to FIG. 30. A chip 1250 has two probe regions corresponding tothe different halves of the shift reticle of FIG. 30. A first region1252 has eight different sets of four probes where the number below(1-8) indicates the position of the interrogation position in theprobes. As before, a "1" indicates that interrogation position is nearerthe chip in the probes, whereas an "8" indicates that the interrogationposition is farther from the chip in the probes.

A second region 1254 has eight different sets of four probes, but asindicated by the number below (1-7), there are two sets of probes withan interrogation position at the fifth base in the probes. The duplicateset of probes was generated because of a blank position in the shiftreticle. Additionally, the probes in region 1254 will be 7-mers andinclude probes with interrogation positions at each possible position inthe probes. Therefore, probes of different lengths and differentinterrogation positions may be synthesized on a chip at the same timewith an embodiment of the shift reticles of the invention.

The formation of duplicate sets of probes may be also utilized toisolate problems during synthesis and/or to increase the accuracy of theresulting data. For example, although the two sets of probes in region1254 that have an interrogation position at the fifth base may beidentical in terms of sequence, the bases were synthesized duringdifferent monomer addition steps. Accordingly, if the fourth monomeraddition step that adds an A is faulty, this may affect one set ofprobes but not the other. Therefore, by analyzing the accuracy of thedata from the duplicate set of probes, one can identify synthesisproblems and since there may be duplicate sets of probes, the synthesisproblems may be accounted for by utilizing another probe set.

In some embodiments, blank probes are placed in the shift reticles atvarious locations so that duplicate probe sets will be formed. Asdiscussed above, the duplicate probe sets may be utilized to isolateproblems during synthesis and possibly even accounting for the errors.

The shift reticles may also be longer to synthesize probes thatinterrogate multiple base positions in the target. FIG. 32 shows a shiftreticle for producing probes that interrogate every ninth base positionin the target. The first part of the shift reticle is the same as shownin FIG. 28 (Reticle 1). However, this shift reticle is longer and may beutilized to interrogate the base positions indicated by the asterisks1275. Probes similar to the one in FIG. 32 may be utilized to form,e.g., 8-mer probes that interrogate every ninth position in the targetwith probes that have every possible interrogation position. Althoughthe interrogation positions in the target that are being interrogated isfixed in the design of the shift reticles, other shift reticles may beproduced to interrogate other positions in the target.

One may also reduce the number of probes by utilizing one set of shiftreticles for the even interrogation positions and one set of shiftreticles for the odd interrogation positions. Both sets of probes areutilized and then shifted. In this manner, probes that haveinterrogation positions at every other possible location may besynthesized. Since there are less probes synthesized on the chip, morebase positions in the target may be interrogated on the chip. Althoughtwo sets of shift reticles have been described (one for even positionsand one for odd positions), more sets of shift reticles may be utilized.For example, one may utilize a different set of shift reticles for eachbase position in the target where (base position mod 3=0), (baseposition mod 3=1), and (base position mod 3=2).

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example, whilethe invention is illustrated primarily with regard to the synthesis ofoligonucleotide or RNA, the invention will find application to thesynthesis of many other molecules. Further, while the invention isprimarily illustrated in relation to the fabrication of small numbers ofidentical arrays, the invention may also be applied to situations wherea large number of identical arrays is to be synthesized. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 7                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 14 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (oligonucleotide)                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #    15                                                                       __________________________________________________________________________

What is claimed is:
 1. A method of synthesizing polymers on a substrate,comprising:coupling monomers on the substrate at locations specified bya plurality of shift reticles, each shift reticle for adding a specificmonomer to the substrate and the monomers are selected from the groupconsisting of nucleotides, amino acids and saccharides; shifting theplurality of shift reticles relative to the substrate; and aftershifting the plurality of shift reticles, coupling monomers on thesubstrate at locations specified by the plurality of shift reticles sothat polymers are synthesized on the substrate that include monomers. 2.The method of claim 1, further comprising utilizing each of the shiftreticles to couple a first layer of different types of monomers on thesubstrate.
 3. The method of claim 2, further comprising shifting each ofthe shift reticles relative to the substrate in order to couple a secondlayer of different types of monomers on the substrate.
 4. The method ofclaim 1, wherein each shift reticle has monomer addition regionscorresponding to positions of the specific monomer in a target sequence.5. A method of synthesizing polymers on a substrate, comprising:couplingmonomers on the substrate at locations specified by a shift reticle,wherein the monomers are selected from the group consisting ofnucleotides, amino acids and saccharides; shifting the shift reticlerelative to the substrate; and after shifting the shift reticle,coupling monomers on the substrate at locations specified by the shiftreticle wherein the shift reticle includes monomer addition regionsspecified by

    n*(i-1)+1

wherein n=the number of different types of monomers and i=a position ofa first monomer in a target sequence.
 6. The method of claim 5, whereinthe shift reticle has monomer addition regions specified by

    n*(i-1)+2

wherein n=the number of different types of monomers and i=a position ofa second monomer in the target sequence.
 7. The method of claim 5,further comprising utilizing the shift reticle to couple a first layerof different types of monomers on the substrate at specified locations.8. The method of claim 7, further comprising shifting the shift reticlerelative to the substrate in order to couple a second layer of differenttypes of monomers on the substrate at the specified locations.
 9. Themethod of claim 1, further comprising:coupling monomers on the substrateat locations specified by an interrogation position reticle; shiftingthe interrogation position reticle relative to the substrate; and aftershifting the interrogation position reticle, coupling monomers on thesubstrate at locations specified by the interrogation position reticle.10. The method of claim 9, wherein the interrogation position reticle isshifted in a direction perpendicular to a direction that the pluralityof shift reticles are shifted.
 11. The method of claim 1, wherein theplurality of shift reticles includes a shift reticle with a monomeraddition region corresponding to each monomer in desired polymers exceptfor non-wild type interrogation position monomers.
 12. The method ofclaim 11, further comprising shifting the shift reticle relative to thesubstrate in order to couple each monomer in the desired polymers exceptfor the non-wild type interrogation position monomers.
 13. The method ofclaim 1, wherein the plurality of shift reticles includes a shiftreticle with monomer addition regions corresponding to different lengthsof desired polymers.
 14. The method of claim 13, further comprisingshifting the shift reticle relative to the substrate in order tosynthesize the desired polymers of different lengths.
 15. The method ofclaim 1, wherein the plurality of shift reticles uniformly adds monomersto the substrate at specified locations after a monomer addition cycle.16. The method of claim 15, further comprising the step of specifying acharacteristic of the polymers desired to be synthesized utilizing theplurality of shift reticles.
 17. The method of claim 16, wherein thecharacteristic of the desired polymers is input at synthesis time. 18.The method of claim 16, wherein the characteristic is a length of thedesired polymers.
 19. The method of claim 16, wherein the characteristicis an interrogation position of the desired polymers.
 20. The method ofclaim 15, wherein the characteristic is a monomer addition in order forsynthesizing the desired polymers.
 21. A method of synthesizing polymerson the substrate, comprising:providing at least one reticle, the atleast one reticle for uniformly adding monomers to the substrate atspecified locations for each cycle of monomer addition steps, whereinthe monomers are selected from the group consisting of nucleotides,amino acids and saccharides; specifying a characteristic of the polymersdesired to be synthesized utilizing the at least one reticle; andsynthesizing the desired polymers on the substrate utilizing the atleast one reticle.
 22. The method of claim 21, wherein thecharacteristic of the desired polymers is input at synthesis time. 23.The method of claim 21, wherein the characteristic is a length of thedesired polymers.
 24. The method of claim 21, wherein the characteristicis an interrogation position of the desired polymers.
 25. The method ofclaim 21, wherein the characteristic is a monomer addition order forsynthesizing the desired polymers.